Regulation of major cultural components for designing a cost effective medium to increase δ-endotoxin synthesis by Bacillus thuringiensis

Abstract

The present study was aimed at designing a cost effective medium for increasing the δ-endotoxin (Cry protein) synthesis by Bacillus thuringiensis (Bt) kurstaki HD-73 and indigenous Bt JSc1 harboring potential cry genes active against Lepidoptera insect orders. In this regard, locally available cheap molasses as carbon source, soybean extract as nitrogen source, sea water as trace elements source, cystine as sporulation/growth factor were considered to design a cost effective medium. Molasses and soybean extract in place of glucose and peptone in glucose-peptone (GP) medium supported 78.85% increase in δ-endotoxin synthesis in shake flask culture. The effect of cystine on endotoxin synthesis was highly pronounced in two media with a range of 80.32 to 110% higher δ-endotoxin under comparable fermentation conditions. While, substituting basal salts with sea water, Btk HD-73 yielded satisfactory and comparable endotoxin (74.3% of yield with basal salts). It was detected that the rapid decrease of endotoxin synthesis in the culture after 24 h was due to the degradation by the endogenous protease, synthesized with the progress of fermentation. This degradation of the endotoxin was much better protected (1.23 mg/ml endotoxin versus 0.312 mg/ml) by adding 4% ammonium sulfate in the optimized medium. The medium thus formulated with molasses, soybean extract, ammonium sulfate, cystine and sea water was then used in 3.0 L bioreactor cultivation for endotoxin synthesis by both Btk HD-73 and Bt JSc1 under 30% saturation of dO₂ through cascade control of agitation and aeration producing  a higher yield of δ-endotoxin (2.1 and 2.63 mg/ml, respectively). The present results may successfully be used for large scale production of Bt biopesticide in Bangladesh.

Keywords: Bacillus thuringiensis biopesticide, cheap substrates, cystine, protease activity, ammonium sulfate.